Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction.

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

Curcumin Derivatives as Potential Mosquito Larvicidal Agents against Two Mosquito Vectors, Culex pipiens and Aedes albopictus.

Vector-borne diseases have appeared or re-emerged in many Southern Europe countries making the transmission of infectious diseases by mosquitoes (vectors) one of the greatest worldwide health threats. Larvicides have been used extensively for the control of Aedes (Stegomyia) albopictus (Skuse, 1895) (Diptera: Culicidae) and Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) mosquitoes in urban and semi-urban environments, causing the increasing resistance of mosquitoes to commercial insecticides. In this study, 27 curcuminoids and monocarbonyl curcumin derivatives were synthesised and evaluated as potential larvicidal agents against Cx. pipiens and Ae. albopictus. Most of the compounds were more effective against larvae of both mosquito species. Four of the tested compounds, curcumin, demethoxycurcumin, curcumin-BF2 complex and a monocarbonyl tetramethoxy curcumin derivative exhibited high activity against both species. In Cx. pipiens the recorded LC50 values were 6.0, 9.4, 5.0 and 32.5 ppm, respectively, whereas in Ae. albopictus they exhibited LC50 values of 9.2, 36.0, 5.5 and 23.6 ppm, respectively. No conclusive structure activity relationship was evident from the results and the variety of descriptors values generated in silico provided some insight to this end.

Isolation of Volatile Compounds with Repellent Properties against Aedes albopictus (Diptera: Culicidae) Using CPC Technology.

The present work describes the use of Centrifugal Partition Chromatography (CPC) for the bio-guided isolation of repellent active volatile compounds from essential oils. Five essential oils (EOs) obtained from three Pinus and two Juniperus species were initially analyzed by gas chromatography-mass spectrometry (GC/MS) and evaluated for their repellent properties against Aedes albopictus. The essential oil from needles of P. pinea (PPI) presented the higher activity, showing 82.4% repellency at a dose of 0.2 µL/cm2. The above EO, together with the EO from the fruits of J. oxycedrus subsp. deltoides (JOX), were further analyzed by CPC using the biphasic system n-Heptane/ACN/BuOH in ratio 1.6/1.6/0.2 (v/v/v). The analysis of PPI essential oil resulted in the recovery of (-)-limonene, guaiol and simple mixtures of (-)-limonene/ß-pheladrene, while the fractionation of JOX EO led to the recovery of ß-myrcene, germacrene-D, and mixtures of α-pinene/ß-pinene (ratio 70/30) and α-pinene/germacrene D (ratio 65/45). All isolated compounds and recovered mixtures were tested for their repellent activity. From them, (-)-limonene, guaiol, germacrene-D as well the mixtures of (-)-limonene/ß-pheladrene presented significant repellent activity (>97% repellency) against Ae. albopictus. The present methodology could be a valuable tool in the effort to develop potent mosquito repellents which are environmentally friendly.

Chemical composition and assessment of larvicidal and repellent capacity of 14 Lamiaceae essential oils against Aedes albopictus.

In the current laboratory study, 14 essential oils (EOs) derived from 12 Lamiaceae plant species and their major components were screened for their larvicidal and repellent properties against Aedes albopictus, an invasive mosquito species of great medical importance. The results of toxicity bioassays revealed that the EOs from Thymus vulgaris, Ocimum basilicum, Origanum dictamnus, Origanum majorana, and Origanum vulgare, as well as their major components (terpenes), namely thymol, carvacrol, p-cymene, and γ-terpinene exerted the highest larvicidal effect. Essential oils from Mellisa officinalis, Origanum dictamus, Mentha spicata (chem. piperitenone epoxide), Origanum majorana, and Satureja thymbra were the most potent repellents, with the last two assigned as the best ones. Among the terpenes tested, piperitenone epoxide, carvacrol, thymol, and piperitenone provided the highest level of protection against Ae. albopictus adults. Chemical analysis revealed the presence of a high number of terpenes in the EOs, while in most cases, the biological action of the tested EOs and their major components was in consistency. The most effective EOs and terpenes that were identified through the current laboratory bioassays could be used as alternative agents to control larvae and repel adults of Ae. albopictus.

Overwintering West Nile virus in active Culex pipiens mosquito populations in Greece.

The flavivirus West Nile Virus (WNV), which is transmitted by mosquitoes, poses a significant threat to both humans and animals, and its outbreaks often challenge public health in Europe and other continents. In recent years, there is an increasing trend of WNV incidence rates across several European countries. However, whether there is a year-round circulation or seasonal introduction has yet to be elucidated. Real-time polymerase chain reaction (PCR) identified WNV-positive Culex pipiens mosquitos in 6 out of 146 pools examined in winter 2022 that correspond to three out of the 24 study areas, located in two coastal regions units in Attica, Greece. Spatial dispersion of the six positive pools in the same region suggests a clustered circulation of WNV during the winter of 2022. This is the first study that documents the identification of WNV in Cx. pipiens populations, captured in adult traps during winter period. Our findings underscore the need to extend entomological surveillance programs to include the winter period, specifically in temperate climates and historically affected areas by WNV.

Sterile Insect Technique (SIT) field trial targeting the suppression of Aedes albopictus in Greece.

The sterile insect technique (SIT) involves releasing large numbers of sterile males to outcompete wild males in mating with females, leading to a decline in pest populations. In the current study, we conducted a suppression trial in Greece against the invasive dengue vector mosquito Aedes albopictus (Skuse) through the weekly release of sterile males for 22 weeks from June to September 2019. Our approach included the long-distance transport of sterile mosquitoes, and their release at a density of 2,547 ± 159 sterile males per hectare per week as part of an area-wide integrated pest management strategy (AW-IPM). The repeated releases of sterile males resulted in a gradual reduction in egg density, reaching 78% from mid-June to early September. This reduction remained between 70% and 78% for four weeks after the end of the releases. Additionally, in the SIT intervention area, the ovitrap index, representing the percentage of traps containing eggs, remained lower throughout the trial than in the control area. This trial represents a significant advance in the field of mosquito control, as it explores the viability and efficacy of producing and transporting sterile males from a distant facility to the release area. Our results provide valuable insights for future SIT programmes targeting Ae. Albopictus, and the methodology we employed can serve as a starting point for developing more refined and effective release protocols, including the transportation of sterile males over long distances from production units to intervention areas.

Title: Essai sur le terrain de la Technique de l'Insecte Stérile (TIS) ciblant la suppression d'Aedes albopictus en Grèce. Abstract: La technique de l'insecte stérile (TIS) consiste à libérer un grand nombre de mâles stériles pour supplanter les mâles sauvages lors de l'accouplement avec les femelles, entraînant ainsi un déclin des populations de nuisibles. Dans la présente étude, nous avons mené un essai de suppression en Grèce contre le moustique vecteur invasif de la dengue, Aedes albopictus (Skuse), par le biais de la libération hebdomadaire de mâles stériles pendant 22 semaines de juin à septembre 2019. Notre approche comprenait le transport sur de longues distances de moustiques stériles, et leur lâcher à une densité de 2 547 ± 159 mâles stériles par hectare et par semaine dans le cadre d'une stratégie de lutte intégrée contre les nuisibles à l'échelle de la zone (AW-IPM). Les lâchers répétés de mâles stériles ont entraîné une réduction progressive de la densité des œufs, atteignant 78 % de la mi-juin au début septembre. Cette réduction est restée entre 70 % et 78 % pendant quatre semaines après la fin des lâchers. De plus, dans la zone d'intervention de la TIS, l'indice d'oviposition, représentant le pourcentage de pièges contenant des œufs, est resté plus faible que dans la zone témoin tout au long de l'essai. Cet essai représente une avancée significative dans le domaine de la lutte contre les moustiques, car il explore la viabilité et l'efficacité de la production et du transport de mâles stériles depuis une installation éloignée vers la zone de lâcher. Nos résultats fournissent des informations précieuses pour les futurs programmes de TIS ciblant Ae. albopictus et la méthodologie que nous avons utilisée pourra servir de point de départ pour développer des protocoles de libération plus raffinés et plus efficaces, y compris le transport de mâles stériles sur de longues distances depuis les unités de production jusqu'aux zones d'intervention.

Essential oil composition, adult repellency and larvicidal activity of eight Cupressaceae species from Greece against Aedes albopictus (Diptera: Culicidae).

The present study evaluated leaf essential oils from eight Cupresaceae species; Cupressus arizonica, Cupressus benthamii, Cupressus macrocarpa, Cupressus sempervirens, Cupressus torulosa, Chamaecyparis lawsoniana, Juniperus phoenicea, and Tetraclinis articulata for their larvicidal and repellent properties against Aedes albopictus, a mosquito of great ecological and medical importance. Based on the LC(50) values, C. benthamii essential oil was the most active (LC(50) = 37.5 mg/L) while the other tested Cupressaceae essential oils provided rather moderate toxicity against larvae (LC(50) = 47.9 to 70.6 mg/L). Under the used laboratory conditions, three of the essential oils (C. benthamii, C. lawsoniana, and C. macrocarpa) provided sufficient protection against mosquito adults, equivalent to the standard repellent "Deet" in the 0.2 mg/cm(2) dose, while C. macrocarpa assigned as the superior repellent oil in the 0.08 mg/cm(2) dose. Chemical analysis of the essential oils using gas chromatography and gas chromatography-mass spectrometry revealed the presence of 125 components.

Profiles of Volatiles Emitted from Orange Fruits Infested by Ceratitis capitata (Diptera: Tephritidae).

The profiles of volatile organic compounds (VOCs) emitted from orange fruits infested by Ceratitis capitata were examined. VOCs were collected from the headspace of oranges immediately, 24 h, 5 days, 8 days, and 13 days after oviposition and when larvae exit the fruit by applying either static or dynamic sampling techniques. A total of 32 and 47 compounds were detected in infested orange fruits when using static and dynamic techniques, respectively. Differences in the volatile profile of oranges were observed. Classification models were employed, showing that infested fruits emitted a chemical profile distinct from that of non-infested ones. Limonene was associated with cell disruption. (E)-ß-Ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene, hexyl butanoate, butyl hexanoate, and hexyl hexanoate were the VOCs that were correlated with the infestation of sweet oranges by C. capitata.

Insights into the Diversity and Population Structure of Predominant Typhlocybinae Species Existing in Vineyards in Greece.

Insects of the subfamily Typhlocybinae (Hemiptera: Cicadellidae) are pests of economically important agricultural and horticultural crops. They damage the plants directly or indirectly by transmitting plant pathogens, resulting in significant yield loss. Several leafhoppers of this subfamily use vines as hosts. Accurate and rapid identification is the key to their successful management. The aim of this study is to determine the Typhlocybinae species that exist in vineyards all over Greece and investigate the relationship between them. For this purpose, yellow sticky traps were placed, morphological and molecular data were collected, and phylogenetic models were analyzed. The mitochondrial marker Cytochrome Oxidase Subunit I (mtCOI) was applied for the DNA and phylogenetic analysis. The combination of morphological and molecular data resulted in identifying the existence of six different species all over Greece: Arboridia adanae, Asymmetrasca decedens, Hebata decipiens, Hebata vitis, Jacobiasca lybica and Zygina rhamni. Forty-eight different haplotypes were found to exist in the different regions of the country.

Comparison of Three Attractants for the Effective Capture of Xylotrechus chinensis Adults in Multi-Funnel Traps.

The Asian coleopteran Xylotrechus chinensis (Chevrolat) (Cerambycidae: Cerambycinae) is an invasive species in several European countries, attacking mulberry trees. In the current research, we evaluated the performance of three mixtures consisting of pheromones and attractants for the monitoring of X. chinensis adults. Attractant 1 (i.e., geranyl acetone, fuscumol acetate, fuscumol, monochamol, 3-hydroxyhexan-2-one, 2-methyl-1-butanol, anti-2,3-hexanediol, prionic acid + ethanol), attractant 2 (i.e., geranyl acetone, fuscumol acetate, fuscumol, monochamol, 3-hydroxyhexan-2-one, 2-methyl-1-butanol, anti-2,3-hexanediol, prionic acid + α-pinene + ethanol) and attractant 3 (i.e., geranyl acetone, fuscumol acetate, fuscumol, monochamol, 3-hydroxyhexan-2-one, 2-methyl-1-butanol, anti-2,3-hexanediol, prionic acid + α-pinene + ipsenol + ethanol) were baited in multi-funnel traps and installed in mulberries for a two-year period in Athens (Greece). The flight activity of X. chinensis starts at the end of April and terminates at the end of October. The peaks of X. chinensis flight activity were observed on 16 August 2021 and on 6 July 2022. Attractant 3 proved to be the most effective blend, catching 953 adults, followed by attractant 2 (523 adults) and attractant 1 (169 adults), throughout the experimental period. It seems that the pest was not attracted to the basic part of the blend (i.e., pheromones + ethanol). The incorporation of α-pinene and ipsenol resulted in the elevated activity of the base lure. The elevated performance of attractant 3 may be attributed to only the α-pinene and the ipsenol, or possibly the α-pinene, ipsenol, and ethanol, because the pheromone blend did not contain any of the pheromone components of the target species. Overall, attractant 3 could be a useful tool to detect and track X. chinensis in new invasive areas, triggering early management strategies against further establishment of this species.

Identification of volatile compounds from fruits aroma and citrus essential oils and their effect on oviposition of Ceratitis capitata (Diptera: Tephritidae).

Ceratitis capitata (Wiedemann), also known as the Mediterranean fruit fly, is one of the most serious pests for several fresh fruit commodities causing extensive fruit losses worldwide. The response of C. capitata adults to fruit and nonfruit volatiles has been extensively studied. However, the linkage between fruit volatiles and the female ovipositional choice has not been fully elucidated. The present study focused on identifying the volatile organic compounds emitted by detached intact fresh fruits (oranges, lemons, bergamots, and apples) and citrus essential oils and evaluate their effect on Mediterranean fruit fly oviposition. There were more than 130 and 45 volatiles compounds detected in fruits odors and citrus essential oils, respectively. The volatile profile of fruits was dominated either from terpenes and terpenoids or from esters of butanoic, hexanoic, and octanoic acids while limonene was by far the most abundant compound in all citrus essential oils. Oviposition of C. capitata was strongly affected by volatiles emanated from both intact fruit and the citrus essential oils. Regarding the volatiles of the intact fruits, the odor of sweet orange elicited strong oviposition responses to females, while bergamot had the least stimulatory effect on oviposition. Bergamot oil also elicited the least oviposition stimulation compared to sweet orange and lemon essential oils. Our discussion elaborates on the role of fruit volatiles on host finding behavior and fruit susceptibility to C. capitata infestation and includes possible practical implication of the above findings.

2D finger-printing and molecular docking studies identified potent mosquito repellents targeting odorant binding protein 1.

Personal protection measures against the mosquitoes like the use of repellents constitute valuable tools in the effort to prevent the transmission of vector-borne diseases. Therefore, the discovery of novel repellent molecules which will be effective at lower concentrations and provide a longer duration of protection remains an urgent need. Mosquito Odorant-Binding Proteins (OBPs) involved in the initial steps of the olfactory signal transduction cascade have been recognized not only as passive carriers of odors and pheromones but also as the first molecular filter to discriminate semiochemicals, hence serving as molecular targets for the design of novel pest control agents. Among the three-dimensional structures of mosquito OBPs solved in the last decades, the OBP1 complexes with known repellents have been widely used as reference structures in docking analysis and molecular dynamics simulation studies for the structure-based discovery of new molecules with repellent activity. Herein, ten compounds known to be active against mosquitoes and/or displaying a binding affinity for Anopheles gambiae AgamOBP1 were used as queries in an in silico screening of over 96 million chemical samples in order to detect molecules with structural similarity. Further filtering of the acquired hits on the basis of toxicity, vapor pressure, and commercial availability resulted in 120 unique molecules that were subjected to molecular docking studies against OBP1. For seventeen potential OBP1-binders, the free energy of binding (FEB) and mode of interaction with the protein were further estimated by molecular docking simulations leading to the selection of eight molecules exhibiting the highest similarity with their parental compounds and favorable energy values. The in vitro determination of their binding affinity to AgamOBP1 and the evaluation of their repellent activity against female Aedes albopictus mosquitoes revealed that our combined ligand similarity screening and OBP1 structure-based molecular docking successfully detected three molecules with enhanced repellent properties. A novel DEET-like repellent with lower volatility (8.55 × 10-4 mmHg) but a higher binding affinity for OBP1 than DEET (1.35 × 10-3 mmHg). A highly active repellent molecule that is predicted to bind to the secondary Icaridin (sIC)-binding site of OBP1 with higher affinity than to the DEET-site and, therefore, represents a new scaffold to be exploited for the discovery of binders targeting multiple OBP sites. Finally, a third potent repellent exhibiting a high degree of volatility was found to be a strong DEET-site binder of OBP1 that could be used in slow-release formulations.

Evaluation of bioefficacy of three Citrus essential oils against the dengue vector Aedes albopictus (Diptera: Culicidae) in correlation to their components enantiomeric distribution.

Laboratory experiments were conducted to study the bioefficacy against Ae. albopictus of three Citrus essential oils, derived from peels of Citrus sinensis, Citrus limon, and Citrus paradise and of their components. Chiral gas chromatography analysis revealed the dominant occurrence of R-(+)-limonene and (-)-ß-pinene in all three essential oils while in the case of lemon oil γ-terpinene, neral, and geranial detected also among other components. The tested Citrus essential oils were toxic against mosquito larvae with LC(50) values ranging from 25.03 to 37.03 mg l(-1). Among citrus essential oils components tested, γ-terpinene was the most toxic (LC(50) = 20.21 mg l(-1)) followed by both enantiomeric forms of limonene (LC(50) = 35.99 and 34.89 mg l(-1), for R-(+)-limonene and S-(-)-limonene, respectively). The delayed toxic effects after exposure of larvae to sublethal (LC(50)) doses were also investigated for citrus essential oils and their major component R-(+)-limonene, indicating a significant reduction of pupal survival. In repellent bioassays, lemon essential oil, S-(-)-limonene, citral (mixture of neral\geranial) and (+)-ß-pinene were the most effective compared with other citrus essential oils and components against adult mosquitoes. Repellent bioassays also revealed that limonenes and ß-pinenes showed an isomer dependence repellent activity. Finally, according to enantiomeric distribution of limonene and α- and ß-pinene, the repellency of lemon essential oil is possibly attributed to the presence of citral.

Colupulone, colupone and novel deoxycohumulone geranyl analogues as larvicidal agents against Culex pipiens.

BACKGROUND: As climate change proceeds, the management of the population of mosquitoes becomes more and more challenging. Insect adulticides and larvicides constitute significant control techniques, with the latter being considered the leading mosquito control method. However, the development of mosquito resistance development and the adverse side effects caused by the extensive use of synthetic insecticides have turned research towards the discovery of environmentally-friendly solutions. Plants and bacteria have historically proven to be a good source of insecticidally active compounds, which may possess novel modes of action to overcome current resistance mechanisms and could also possess favorable human and environmental safety profiles. A previous study demonstrated that the naturally occurring prenylated acyl phloroglucinol deoxycohumulone is a potent larvicidal agent against Culex pipiens. Herein the structural characteristics that improve it are explored by evaluating colupulone and novel geranylated analogues. RESULTS: Colupulone, a prenylated acyl phloroglucinol isolated from Humulus lupulus, colupone, and novel geranylated acyl phloroglucinol congeners, were synthesized and evaluated against Cx. pipiens larva. Results indicated that selected derivatives exhibited superior potency than deoxycohumulone (LC50 43.7 mg L-1 ). Thus, strong activity was observed for colupulone (LC50 19.7 mg L-1 ), and some novel geranyl analogues of deoxycohumulone reaching at LC50 17.1 mg L-1 , while colupone and similar compounds were almost inactive. CONCLUSION: The results determined the relationship between the target activity and the chemical structure of the tested compounds, and they revealed significantly improved larvicidal candidates. These results highlight the potential of the acyl phloroglucinol chemistry for further development of mosquito larvicides. © 2022 Society of Chemical Industry.

Behavioural response of Culex pipiens biotype molestus to oviposition pheromone.

Insect behaviour relies on an olfactory sensory system that controls a range of activities, from food choice and mating to oviposition, where pheromones play a central role. In Culex mosquitoes, egg-laying is accompanied by the release of mosquito oviposition pheromone (MOP), which has been shown to affect the oviposition behaviour of conspecifics. Here, we investigated for the first time the effect of MOP on the oviposition rate of Culex pipiens biotype molestus, examining separately males and females, before and after mating and oviposition. Our results demonstrate that MOP is more likely to act as an oviposition stimulant rather than an attractant, since more gravid females laid eggs in its presence, while the number of male or female mosquitoes (virgin or mated) captured on pheromone-treated pots was similar to those treated with control water.

A Molecular Diagnostic Assay for the Discrimination of Aphid Species (Hemiptera: Aphididae) Infesting Citrus.

Aphid species (Hemiptera: Aphididae) are among the most serious pests for citrus cultivation throughout the world causing substantial crop damages. Accurate identification of aphids to the species level can be difficult, though being crucial for their effective management. In this study, a molecular diagnostic assay for distinguishing eleven aphid species was developed. A fragment of the mitochondrial Cytochrome Oxidase I (mtCOI) gene was used and a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-FLP) analysis with five restriction enzymes, based on DNA sequence polymorphisms, was applied to differentiate the eleven aphid species. This molecular technique allows aphid species at any life stage to be discriminated accurately and simply and can be a useful tool for monitoring the populations of economically important aphid species.

Knowledge, Attitudes and Perception of Mosquito Control in Different Citizenship Regimes within and Surrounding the Malakasa Open Accommodation Refugee Camp in Athens, Greece.

The study aims to evaluate the Knowledge, Attitude and Perception (KAP) of different societal groups concerning the implementation of targeted community-based mosquito surveillance and control interventions in different citizenship regimes. Targeted surveys were carried out within Malakasa camp for migrants and refugees, neighboring residential areas and urban areas in the wider Athens metropolitan area to investigate different knowledge levels and the role that both local and migrant communities can play in the implementation of community-based interventions based on their attitudes and perceptions. A scoring system was used to rate the collected responses. Results indicate different levels of KAP among the various groups of respondents and different priorities that should be considered in the design and execution of community interventions. Findings indicate a lower level of Knowledge Attitudes and Perceptions for the migrants, while the rate of correct answers for Perception significantly improved for migrants following a small-scale information session. The study highlights disparities in the levels of knowledge for certain public health issues and the feasibility of certain approaches for alleviating health-related challenges such as mosquito-borne diseases. Findings suggest that essential preparedness is needed by public authorities to respond to public health challenges related to migration and the spread of vector-borne diseases.

Quality Control Methods for Aedes albopictus Sterile Male Transportation.

Genetic based mosquito control methods have been gaining ground in recent years for their potential to achieve effective suppression or replacement of vector populations without hampering environments or causing any public health risk. These methods require the mass rearing of the target species in large facilities sized to produce millions of sterile males, as already well established for a number of insects of agricultural importance. Assessing the performance of released males in Sterile Insect Technique (SIT) control programs is of the utmost importance for the success of the operation. Besides the negative effects of mass rearing and sterilization, the handling of sterilized insects and shipment to distant areas may also negatively impact the quality of sterilized males. The aim of the current study was to design and executive quality control (QC) tests for sterilized Aedes albopictus (Asian tiger mosquito) males delivered by air shipment from the mass production facility located in Italy to Greece and Montenegro field release sites. Mass reared mosquito strains were based on biological materials received from Italy, Greece and Montenegro. Tests conducted at the mass rearing facility before transportation revealed a rather high residual female contamination following mechanical sex separation (approximately 1.5% females, regardless of the mosquito strain). Irradiated males of all three mosquito strains induced high levels of sterility to females. Shipment lasting approximately 24 h resulted in approximately 15% mortality, while when shipment lasted nearly two days this increased to almost 40%. The flight ability of sterilized males following one day transportation time was satisfactory (over 60%). The response of sterile males to food and water starvation was comparable and slightly lower than that of wild non-transported males. Longevity of sterile males was shorter than that of wild counterparts and it seems it was not affected by mating to wild females. Both mating propensity and mating competitiveness for wild virgin females was higher for the wild, control males compared to the sterile, transported ones. Overall, the performance of sterile male Ae. albopictus delivered from the mass rearing facility of Italy to Greece in approximately 24 h was satisfactory. Transportation lasting two days or longer incurred detrimental effects on males, which called into question the outcome of the SIT release programs. In conclusion, our results demonstrate the need of quality control procedures, especially when sterile male production facilities are not near to the releasing point. Transportation could be a serious drawback for the implementation of Sterile Insect Releases and, consequently, it is important to establish an efficient and fast transportation of sterilized males in advance.

Efficacy Evaluation of Oregano Essential Oil Mixed with Bacillus thuringiensis israelensis and Diflubenzuron against Culex pipiens and Aedes albopictus in Road Drains of Italy.

Mosquito management programs in the urban environment of Italian cities mainly rely on larval control with conventional insecticides, primarily targeting the road drains that constitute the principal mosquito breeding sites encountered in public. The repeated utilization of synthetic insecticides may have adverse effects on non-targets and lead to resistance development issues, while the performance of biopesticides encounters limitations in field use. Botanical insecticides as single larval control agents or in binary mixtures with conventional insecticides have been extensively studied in the laboratory as an effective and eco-friendly alternative mosquito control method with promising results. The study herein concerns the investigation, for the first time under realistic conditions in the field, of the joint action of a carvacrol-rich oregano Essential Oil (EO) with two conventional insecticides, namely, the insect growth regulator diflubenzuron and the bio-insecticide Bacillus thuringiensis israelensis (B.t.i.), in road drains in Crevalcore city, Italy, against Culex pipiens and Aedes albopictus. According to the obtained results, the application of both plain EO and its mixtures with diflubenzuron and B.t.i. exerted very high efficacy in terms of immature mosquito population reduction over a two-week period. Three weeks after treatment, the performance of the oil and its mixtures diminished but remained high, while the addition of diflubenzuron potentiated the persistent action of the oil against Cx. pipiens. These findings are indicative of the potential of mixing carvacrol-rich EO with diflubenzuron and B.t.i. as an efficient eco-friendly alternative to mono-insecticide applications in road drains against Cx. pipiens and Ae. albopictus larvae.

Seasonal Appearance, Abundance, and Host Preference of Philaenus spumarius and Neophilaenus campestris (Hemiptera: Aphrophoridae) in Olive Groves in Greece.

Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae) is a xylem inhabiting bacterium which is exclusively transmitted by xylem sap feeding insects. Among them, Philaenus spumarius Linnaeus and Neophilaenus campestris Fallén are the most abundant species in Europe. During 2018 and 2019 a survey was conducted in olive groves in Greece aimed to improve the knowledge about the biology and ecology of those spittlebugs in areas with Mediterranean climate. Moreover, the host preference of the nymphs was studied. The nymphs of P. spumarius and N. campestris were observed between early March and middle May depending on geographic location and year. The spittlebug adults were present during two periods every year, one in spring and another one in autumn and early winter. During summer months the spittlebugs were totally absent from olive groves. Our observation on host plant selection revealed that there was a strong preference of N. campestris nymphs for plants belonging to the family Poaceae. On the contrary, P. spumarius nymphs were polyphagous. The most preferable plants for the nymphs of this species belonged to the families Asteraceae and Fabaceae. The importance of these findings for control measures for these spittlebugs is discussed.